Project Summary EZH2 (enhancer of zeste homolog 2) is the main enzymatic subunit of PRC2 (polycomb repressive complex 2) that catalyzes methylation of histone H3 lysine27 (H3K27). EZH2 is overexpressed in a broad spectrum of cancers including triple-negative breast cancer (TNBC) and high expression correlates with poor prognosis. However, EZH2 inhibitors are ineffective at inhibiting proliferation of breast cancer cell lines with EZH2 overexpression including TNBC cell lines even though they effectively inhibit the enzymatic activity of EZH2/PRC2. It has also been shown that knockdown of EZH2 significantly blocks growth of breast cancer cell lines including TNBC cells. Together, these results suggest that overexpression of EZH2, but not the methyltransferase activity of EZH2, is critical for driving breast cancer cell proliferation. We therefore hypothesized that EZH2 degraders, which refer to small molecules that selectively reduce EZH2 protein levels, would be effective therapeutics for treating TNBC and other subtypes of breast cancer with EZH2 overexpression. To test this therapeutic hypothesis, we propose to develop EZH2 degraders by using the bivalent inhibitor technologies such as PROTACs (proteolysis targeting chimeras) and hydrophobic tagging, which have been successfully applied to selective degradation of multiple protein targets. During our preliminary studies, we have generated first-in-class EZH2 degraders using the bivalent inhibitor technologies. Our prototype EZH2 degraders significantly reduced EZH2 protein levels and displayed robust antiproliferative effects in TNBC cellular and xenograft mouse models. Based on the promising preliminary results, we propose to: (1) optimize EZH2 degraders so that they have in vitro and cellular potency, selectivity, and pharmacokinetic properties consistent with an in vivo chemical probe, and (2) evaluate improved EZH2 degraders in TNBC cellular and mouse models. We expect that the proposed research will validate our hypothesis that pharmacological degradation of EZH2 would provide a novel therapeutic strategy for treating TNBC. The chemical probes generated here can be further optimized into a drug candidate for translating in the clinic for TNBC patients. These EZH2 degraders are also valuable chemical tools for assessing the potential of EZH2 degradation in other cancers such as prostate cancer, lymphoma, myeloma and other subtypes of breast cancer with EZH2 overexpression.